A 3d liver chip that can assess the risk of drug-induced liver injury mediated by transporters

Abstract

The present application belongs to the field of biomedical engineering, microfluidic technology and drug toxicity evaluation, and relates to a 3D liver chip capable of evaluating the risk of drug-induced liver injury mediated by transporters. Specifically, the present application provides a liver chip platform based on dynamic three-dimensional (3D) cell culture with transporter phenotype characteristics, and its application in evaluating and predicting the risk of drug-induced liver injury (DILI) related to liver drug transporters.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering, microfluidics technology and drug toxicology assessment, and specifically relates to a 3D liver chip that can assess the risk of transporter-mediated drug-induced liver injury. Background Technology

[0002] Drug-induced liver injury (DILI) associated with cholestasis accounts for approximately 40% of reported DILI-related development terminations and drug withdrawals. In the process of drug-induced cholestasis, changes in the function of the hepatobiliary transporter system are considered to be the main inducing factor, and efflux transporters on the bile duct lateral membrane of hepatocytes are closely related to cholestatic DILI.

[0003] The liver processes up to 30-40 grams of bile salts daily. Bile salts are powerful detergents that dissolve fats, but they also possess inherent cytotoxicity—they can dissolve cell membranes, damage mitochondria, and induce apoptosis. Hepatocytes survive this high-concentration bile salt environment thanks to a sophisticated network of transport proteins that maintains extremely low levels of bile salts in the cytoplasm. This network is known as the "hepatic transporter." When drugs interfere with these transporters, alterations in their function can disrupt various endogenous and exogenous processes in the body, leading to drug-induced liver injury (DILI), including drug-drug interactions, cholestatic liver injury, and mixed liver injury.

[0004] Therefore, it is essential to develop suitable and reliable liver models for screening and assessing the risk of hepatotoxicity related to hepatic drug transporters in preclinical studies of new drugs. Summary of the Invention

[0005] This invention provides a method for establishing a dynamic 3D liver microarray platform that can simulate the function of transporters in hepatocytes and possess transporter characteristics, as well as its applications. This method is characterized by controllable cost, simple operation, and no need for external inducing agents, providing a practical new platform for research on liver transporter-related mechanisms, drug screening, and toxicity assessment.

[0006] In a first aspect of the invention, a method is provided for safety screening of candidate drugs to obtain potential drugs with hepatoprotective properties, the method comprising the following steps: (S1) A drug-induced liver injury risk assessment device is provided, the device comprising: a microfluidic chip and a power unit; the microfluidic chip comprises: a cell culture chamber and a flow path plate containing perfusion channels; in, When the cell culture chamber is embedded in the flow path plate, the cell culture chamber is located above the perfusion channel, and the cell culture chamber and the perfusion channel are separated by a porous membrane structure; the porous membrane allows liquid exchange between the cell culture chamber and the perfusion channel; The inlet and outlet of the perfusion channel in the flow path plate are connected by an external pipeline connected to a power device. The external pipeline and the perfusion channel inside the microfluidic chip together form a circulation path for flow perfusion culture, thereby realizing flow perfusion culture of cells in the cell culture chamber. in, The cell culture chamber contains a 3D cell structure formed from hepatocytes; The hepatocytes include: liver cancer cells; The levels of liver transport proteins in the hepatocytes were significantly increased; wherein the liver transport proteins include: MRP2, BSEP, or a combination thereof; (S2) In the experimental group, cells were cultured in a cell culture chamber in the presence of the candidate drug; in the control group, cells were cultured in a cell culture chamber under the same conditions but in the absence of the candidate drug. (S3) The concentrations (C1) of hepatic transporter proteins in the cells of the experimental group and (C2) of hepatic transporter proteins in the cells of the control group were detected and compared. If C1 was significantly less than C2, it indicated that the candidate drug had a risk of liver injury; otherwise, it indicated that the candidate drug was a potential drug with hepatoprotective properties; and / or The concentrations of specific substrates of liver transport proteins in the cells of the experimental group (D1) and the concentrations of specific substrates of liver transport proteins in the cells of the control group (D2) were measured and compared. If D1 was significantly greater than D2, it was suggested that the candidate drug had a risk of liver damage; otherwise, it was suggested that the candidate drug was a potential drug with liver safety. The liver injury risk mentioned above refers to the risk of drug-induced liver injury mediated by transporters.

[0007] In another preferred embodiment, "significantly less than" means C1 / C2 ≤ 0.8, more preferably ≤ 0.6, even more preferably ≤ 0.5, further preferably ≤ 0.3, and most preferably ≤ 0.1.

[0008] In another preferred embodiment, "significantly greater than" means D1 / D2 ≥ 2, more preferably ≥ 3, even more preferably ≥ 5, and most preferably ≥ 8.

[0009] In another preferred embodiment, the cell culture chamber contains only one type of hepatocytes.

[0010] In another preferred embodiment, the candidate drug is a small molecule drug.

[0011] In another preferred embodiment, the candidate drug is a single-component substance or a mixture of defined components.

[0012] In another preferred embodiment, the candidate drug comprises: a candidate insulin sensitizer.

[0013] In another preferred embodiment, the liver cancer cells comprise: HepG2 cells, HepRG cells, Huh7 cells, or a combination thereof.

[0014] In another preferred embodiment, the risk of liver injury includes: cholestasis, mitochondrial damage, oxidative stress, endoplasmic reticulum stress, or a combination thereof.

[0015] In another preferred embodiment, the power unit includes a peristaltic pump.

[0016] In another preferred embodiment, the external piping is closed.

[0017] In another preferred embodiment, the external conduit refers to a silicone tube.

[0018] In another preferred embodiment, the 3D structure comprises a 3D cellular spheroid structure.

[0019] In another preferred embodiment, the diameter of the 3D cell spheroid structure is 10-300 µm, more preferably 10-200 μm.

[0020] In another preferred embodiment, the porous membrane has a pore size of 0.4-30 µm.

[0021] In another preferred embodiment, the flow path plate includes one or more irrigation conduits.

[0022] In another preferred embodiment, one or more cell culture chambers are contained above the liquid flow path from the inlet to the outlet in each perfusion channel.

[0023] In another preferred embodiment, the one or more refers to 1-10, more preferably 1-8, more preferably 1-6, and most preferably 2-5.

[0024] In another preferred embodiment, each irrigation channel also includes a reservoir above it.

[0025] In another preferred embodiment, the liquid storage chamber is located at one end near the inlet of the irrigation channel.

[0026] In another preferred embodiment, the fluid shear force generated in the irrigation channel is 0.01-3.0 dyn / cm. 2 The preferred value is 0.5-1.0 dyn / cm. 2 .

[0027] In another preferred embodiment, the cell culture chamber contains a 3D cell structure formed by hepatocytes encapsulated in an extracellular matrix.

[0028] In another preferred embodiment, the extracellular matrix comprises: BME, collagen, hyaluronic acid, Matrigel, or a combination thereof.

[0029] In another preferred embodiment, the circulating liquid in the perfusion culture comprises a culture medium.

[0030] In another preferred embodiment, step (S3) further includes: The mitochondrial membrane potential A1 of the cells in the experimental group and the mitochondrial membrane potential A2 of the cells in the control group were detected and compared. If there was no significant difference between A1 and A2, it would suggest that the candidate drug is a potential drug with liver safety.

[0031] In another preferred embodiment, the lack of significant difference means that A1 / A0 is 0.8-1.2, more preferably 0.9-1.1, and most preferably 0.95-1.05.

[0032] In another preferred embodiment, the mitochondrial membrane potential is represented by a TMRM. + Cell count characterization.

[0033] In another preferred embodiment, step (S3) further includes an evaluation of changes in intracellular ATP content, changes in CYP3A4 enzyme activity, or a combination thereof, compared to the control group.

[0034] In a second aspect of the invention, a microfluidic chip for drug-induced liver injury risk assessment is provided, the microfluidic chip comprising: a cell culture chamber and a flow path plate including perfusion channels; in, When the cell culture chamber is embedded in the flow path plate, the cell culture chamber is located above the perfusion channel, and the cell culture chamber and the perfusion channel are separated by a porous membrane structure; the porous membrane allows liquid exchange between the cell culture chamber and the perfusion channel; The irrigation channel includes an inlet and an outlet; in, The cell culture chamber contains a 3D cell structure formed from hepatocytes; The liver cells include liver cancer cells.

[0035] In another preferred embodiment, the cell chamber includes a sidewall, a top, and a bottom, the sidewall being a closed structure, the top having an opening, and the bottom being composed of a porous membrane.

[0036] In a third aspect of the present invention, a drug-induced liver injury risk assessment device is provided, the device comprising: the microfluidic chip and power device described in the second aspect of the present invention; The inlet and outlet of the irrigation channel in the flow path plate are connected by an external pipeline connected to a power device. The external tubing and the internal perfusion channels of the microfluidic chip together form a circulation path for flow perfusion culture.

[0037] In a fourth aspect of the invention, the use of the microfluidic chip described in the second aspect of the invention or the device described in the third aspect of the invention is provided for: (a) Screening for potential drugs with hepatoprotective properties; and / or (b) Assess the risk of liver injury from candidate drugs in vitro; (c) Differentiate the acute hepatotoxic effects of drugs from the cumulative effects of chronic hepatotoxicity in vitro; (d) Evaluate in vitro the effects of the drug on the expression levels, subcellular localization, and efflux activity of MRP2 and / or BSEP.

[0038] In another preferred embodiment, the use is for non-diagnostic and non-therapeutic purposes.

[0039] In another preferred embodiment, the liver injury includes: acute liver injury and chronic liver injury.

[0040] In a fifth aspect of the present invention, a method for preparing the microfluidic chip described in the second aspect of the present invention is provided, the method comprising the following steps: (I) Provide a cell culture chamber and a flow path plate containing perfusion channels; The irrigation channel includes an inlet and an outlet; When the cell culture chamber is embedded in the flow path plate, the cell culture chamber is located above the perfusion channel, and the cell culture chamber and the perfusion channel are separated by a porous membrane structure; the porous membrane allows liquid exchange between the cell culture chamber and the perfusion channel; (II) Provides a 3D cellular structure formed from hepatocytes, The hepatocytes include: liver cancer cell lines; (III) The 3D cell structure provided in step (II) is transferred to the cell culture chamber provided in step (I) to obtain the microfluidic chip described in the second aspect of the present invention.

[0041] In another preferred embodiment, in step (I), the cell culture chamber has been embedded in the flow path plate.

[0042] In another preferred embodiment, in step (III), after transferring the 3D cell structure provided in step (II) into the cell culture chamber provided in step (I), the cell culture chamber containing the 3D cell structure is embedded into the flow path plate to obtain the microfluidic chip described in the second aspect of the present invention.

[0043] In another preferred embodiment, in step (III), the transfer occurs once or multiple times.

[0044] In another preferred embodiment, "multiple times" refers to 2-3 times.

[0045] In another preferred embodiment, step (II) includes the following sub-steps: (II-1) The hepatocytes were seeded into microplates with low adsorption and cultured to form 3D cell structures; (II-2) Collect the 3D cell structure and disperse it in a premix of extracellular matrix and pre-cooled culture medium to obtain an embedded 3D cell structure, thereby providing a 3D cell structure formed by hepatocytes.

[0046] In another preferred embodiment, the extracellular matrix refers to basement membrane extract (BME).

[0047] In another preferred embodiment, the volume ratio of the culture medium to the extracellular matrix is ​​1:1 to 1:5, more preferably 1:1 to 1:3, and most preferably 1:2.

[0048] In another preferred embodiment, the culture medium comprises: basal culture medium, serum, and matrix gel.

[0049] In another preferred embodiment, the culture medium contains: antibiotics.

[0050] In another preferred embodiment, the antibiotic refers to penicillin and streptomycin.

[0051] In another preferred embodiment, the basal culture medium comprises: DMEM medium, high glucose medium, or optionally a specific hepatocyte cell line medium.

[0052] In another preferred embodiment, the serum is 10% fetal bovine serum.

[0053] In another preferred embodiment, the matrix adhesive comprises: Matrigel, collagen, hyaluronic acid, or a combination thereof.

[0054] In another preferred embodiment, the concentration of the matrix adhesive is 0.5%-5%, more preferably 0.5%-3%, and most preferably 0.5%-1.5%.

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0056] Figure 1 A schematic diagram and workflow of the liver chip platform of the present invention are shown. Figure 1 A shows the front and top views of the chamber used in the 3D dynamic liver chip; Figure 1 B shows the structure of the microfluidic chip used in the dynamic 3D liver chip; Figure 1 C shows the assembly process of the microfluidic system used in the 3D dynamic liver chip; Figure 1 D shows the culture and preparation process of the 3D dynamic liver chip.

[0057] Figure 2 A shows the expression and polarization of MRP2 and BSEP transporters in dynamic 3D liver chip spheroids and 2D monolayer cells, and the comparison of ZO-1 protein expression. Figure 2 B and Figure 2 C shows the differences in the expression levels of MRP2 and BSEP transporters in the 3D hepatocyte sphere 2D monolayer cell culture model, respectively. Figure 2 D and Figure 2 E shows the difference in CDFDA efflux between 3D hepatocyte spheres and 2D cultured monolayer hepatocytes. Figure 2 F shows the differences in mitochondrial function between 3D hepatocyte spheres and 2D cultured monolayer hepatocytes. Figure 2 G shows the difference in ATP function between 3D hepatocyte spheres and 2D cultured monolayer hepatocytes. Figure 2 H shows the difference in CYP3A4 enzyme expression between 3D hepatocyte spheres and 2D cultured monolayer hepatocytes.

[0058] Figure 3 A and Figure 3 B respectively shows the dynamic 3D hepatocyte spheres of the present invention ( Figure 3 A) and 2D hepatocytes ( Figure 3 B) The effect of monolayer treatment with 20, 50, and 100 μM troglitazone on CDFDA efflux. Figure 3 C and Figure 3 D respectively showed Figure 3 3D hepatocyte spheres in A Figure 3 The amount of intracellular CDF in 2D hepatocyte monolayers in B, after treatment with 20, 50, and 100 μM troglitazone, was detected by the CDFDA probe. Figure 3 E shows the relative mitochondrial membrane potential values ​​of dynamic 3D hepatocyte spheres and 2D hepatocyte monolayers after treatment with 20, 50, and 100 μM troglitazone.

[0059] Figure 4 A and Figure 4 B shows fluorescence images of CDFDA in hepatocytes and the relative content of intracellular CDF, obtained by CDFDA probe testing after 24 hours of treatment with different concentrations of omaglione on dynamic 3D hepatocyte spheres. 3D hepatocyte spheres treated with 100 μM troglitazone served as a positive control. Figure 4 C Figure 4 D、 Figure 4 E and Figure 4 F shows the fluorescence images, intracellular CDF concentration, hepatocyte ATP content, and mitochondrial membrane potential detected by the CDFDA probe after 7 days and 2 hours each day of treatment with 10 and 100 μM oxaliplatin. Detailed Implementation

[0060] Through extensive and in-depth research, the inventors have unexpectedly discovered a liver microarray platform based on dynamic three-dimensional (3D) hepatocellular carcinoma cell culture, featuring transporter phenotypic characteristics. This platform, which cultures cell spheres in a dynamically perfused microarray with open culture chambers, simulates the hepatocyte drug transporter system and exhibits hepatic transporter expression characteristics. As a novel in vitro model of hepatic transporters, this microarray platform can not only be used to observe the function and expression characteristics of hepatic transporters but also serve as a drug hepatotoxicity testing model, particularly for assessing cholestatic drug-induced liver injury (DILI) toxicity related to hepatic transporters. Furthermore, it can be combined with various detection methods to study the pathological mechanisms of transporter-mediated hepatotoxicity. This invention offers advantages such as simple operation, standardized reproducibility, no need for inducing factors, wide cell adaptability, low cost, and simple sampling. It forms 3D cell spheres with hepatic transport characteristics, which can replace traditional animal models and simple two-dimensional cell culture models, providing a powerful and reliable testing and evaluation platform for transporter-mediated hepatotoxicity and risk prediction and drug screening for cholestatic DILI. This invention was completed based on this foundation.

[0061] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0062] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0063] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0064] As used in this article, the term "or a combination thereof" means "or any combination thereof".

[0065] As used in this article, the term "3D cell structure" refers to a three-dimensional cell copolymer formed by multiple cells under three-dimensional (3D) cell culture technology.

[0066] As used herein, the term "liver safety" refers to a low risk of liver injury. In specific embodiments, a candidate drug is liver safe if the liver safety results exhibit one or more characteristics selected from the group consisting of: low incidence (incidence ≤1%, preferably ≤0.1%, best ≤0.01%), mild or severe (even if liver injury occurs, it is reversible upon discontinuation of the drug), wide therapeutic window (safe at conventional doses), or a combination thereof. In specific embodiments, a low risk of liver injury means that there is no significant difference in the incidence of liver injury compared to placebo. In specific embodiments, a low risk of liver injury means that there is no significant difference compared to similar clinical drugs or that the candidate drug has a significantly lower risk of liver injury than similar drugs. In specific embodiments, a low risk of liver injury means that the candidate drug does not significantly increase the risk of liver injury compared to the risk of spontaneous liver injury in untreated healthy individuals.

[0067] As used herein, unless otherwise stated, "significant" means statistical significance. In specific embodiments, "significant" is defined according to the clinical medical definition. In specific embodiments, "significant" is defined according to Hay's Law.

[0068] Extravasation transporters on the bile duct lateral membrane of hepatocytes and cholestatic DILI Efflux transporters on the bile duct lateral membrane of hepatocytes are core functional units for maintaining bile flow and preventing cholestatic drug-induced liver injury. The most critical of these is the bile salt export pump (BSEP), which pumps bile salts from within hepatocytes against their concentration gradient into the bile ducts, providing the driving force for bile salt-dependent bile flow. When drugs or metabolites directly inhibit BSEP function, intracellular bile salts accumulate, triggering mitochondrial oxidative stress and apoptosis. This is the molecular basis of progressive familial intrahepatic cholestasis of disease type 2 (FIC) and the initiating event of clinical cholestasis caused by drugs such as troglitazone and bosentan. The multidrug resistance-associated protein MRP2 mediates the efflux of glutathione and non-bile salt organic anions; its inhibition exacerbates efflux impairment on the bile duct lateral side. More severe hepatotoxicity often stems from the synergistic inhibition of BSEP, MRP2, and their compensatory efflux pumps MRP3 / MRP4 by drugs, leading to complete loss of bile salt efflux and safety valve mechanisms within the cells. Therefore, the functional integrity of extravasation transporters in the bile duct lateral membrane is the molecular defense for hepatocytes to survive in the bile salt environment, and its impairment is the central pathogenic mechanism of cholestatic DILI.

[0069] Liver models are crucial for screening and assessing hepatotoxicity risks associated with hepatic drug transporters in preclinical drug research. However, classic small animal models differ significantly from humans in the types and expression of transporters, metabolic enzymes, and other functional proteins, making it difficult to directly extrapolate predictions to humans. Traditional two-dimensional monolayer cell models struggle to obtain and maintain mature hepatocyte phenotypic characteristics, failing to meet the needs for assessing and measuring liver injury beyond direct cellular damage mechanisms. Among in vitro models targeting hepatic transporters, the commonly used membrane-flipped vesicle model lacks a cellular background, can only assess one transporter, limiting its application and hindering further mechanistic and pathological studies. Polarized monolayer sandwich cell models often require lengthy induction processes, making them unsuitable as platforms for drug screening and testing. While organ-on-a-chip and other microphysiological system culture methods combining microfluidics can more accurately reflect and reproduce liver physiological functions and characteristics, and complex in vitro models such as organoids can faithfully simulate in vivo physiological processes, they face limitations such as complex and difficult-to-standardize culture procedures, high reproducibility, and high costs, making them unsuitable as universal tools for drug screening and evaluation.

[0070] Liver-on-a-chip (LIB) is a microphysiological system constructed on transparent materials using microfluidic technology to simulate the smallest functional unit of the liver. Currently, the hepatotoxicity of drugs that have been discontinued or withdrawn from the market due to drug-related hepatotoxicity falls into four main categories: mitochondrial damage, oxidative stress, endoplasmic reticulum stress, and cholestasis. The first three can be evaluated using existing in vitro liver models, primarily focusing on hepatocyte damage, and can be assessed for hepatotoxicity related to mitochondrial damage, oxidative stress, and endoplasmic reticulum stress. However, for cholestatic hepatotoxicity mediated by transporters, existing sandwich-structured cells require the addition of exogenous inducers to promote the model and obtain characteristic hepatocyte phenotypes. Furthermore, the cells need to be adjusted to a calcium-free environment for testing, which can easily deviate from normal cell conditions, leading to questionable results. While liver organoids can be induced to differentiate into more comprehensive hepatocyte types and can be used to evaluate cholestatic hepatotoxicity, this is not the case. However, liver organoids are generally obtained through iPSC-induced differentiation. The maturity of liver organoid spheres, batch-to-batch variations in induction and differentiation, and differences in laboratory procedures result in poor reproducibility of liver organoid physiological models and make it difficult to establish unified evaluation standards. Furthermore, liver-on-a-chip physiological models constructed from primary hepatocytes derived from human liver tissue suffer from the rarity of tissue sources, limiting their large-scale and commercial application. Currently, there is no stable, highly reproducible, standard 3D liver-on-a-chip platform for assessing liver transporter-related toxicity and conducting pathological research. In particular, early risk prediction studies of chronic drug-induced cholestasis hepatotoxicity urgently require a hepatotoxicity evaluation technology platform that combines biomimicry, high reproducibility, and ease of standardized operation, while also eliminating the need for exogenous inducers to exclude factors interfering with drug action, thus supporting testing needs.

[0071] The liver chip platform of the present invention This invention provides a 3D liver chip platform, which is a dynamic 3D liver chip platform, comprising: Microfluidic chip body: The body integrates at least one chamber for cell culture and a chip base with perfusion channels that can be combined with the culture chamber; Porous membrane: disposed at the bottom of the culture chamber, serving as a support interface for the 3D cell sphere construct and a material exchange interface between the chamber and the perfusion channel; Dynamic perfusion system: driven by a pump and connected to the perfusion channel chip base, used to provide a continuous flow of culture medium; 3D cell construct: located within the cell culture chamber, comprising pre-formed 3D cell spheres uniformly encapsulated in the extracellular matrix; The platform, under dynamic perfusion culture, can enhance the 3D cell spheroids and maintain their cell polarity, and highly express the functional hepatic efflux transport proteins MRP2 and BSEP.

[0072] Furthermore, the extracellular matrix is ​​basement membrane extract (BME), Matrigel, or other matrix gels.

[0073] Furthermore, the fluid shear force generated by the dynamic irrigation system is 0.5-1.0 dyn / cm. 2 To simulate the physiological microenvironment of liver cells in vivo.

[0074] In a specific embodiment, the present invention provides a dynamic 3D liver chip platform for assessing the risk of transporter-mediated drug-induced liver injury, comprising: A 3D cell sphere construct comprising 3D cell spheres preformed and uniformly encapsulated in an extracellular matrix, placed within a cell culture chamber of a microfluidic system; The 3D cell sphere construct is placed in the cell culture chamber, which is a cavity structure with a porous membrane at the bottom. The porous membrane is used to separate the culture chamber and the perfusion channel, serving as the cell culture base and the interface for substance exchange. The microfluidic chip body integrates at least one cell culture chamber and a chip base with a perfusion channel; The 3D cell sphere construct is located in a dynamic perfusion system and connected to the chip base of the perfusion channel to provide a continuous flow of culture medium and drug loading. The cell sphere construct in the platform can enhance and maintain cell polarity, and is effective for hepatocytes that highly express functional efflux transporters such as multidrug resistance-associated protein 2 (MRP2) and bile salt export pump (BSEP), as well as uptake transporters such as sodium taurocholic acid cotransport polypeptide (NTCP) and organic anion transport polypeptide OATP1B1.

[0075] In another preferred embodiment, the dynamic irrigation system employs a fluid shear force of 0.01-3.0 dyn / cm2.

[0076] Method for fabricating liver chip platform of the present invention This invention provides a method for constructing the 3D liver chip platform, comprising the following steps: (S1) Preparation of cell spheroids: The cells are seeded in a low-adsorption plate and cultured to allow them to spontaneously aggregate and form uniformly sized cell spheroids; (S2) Chip preparation: Provide the microfluidic chip body, attach a porous membrane to the bottom of the culture chamber, and assemble and combine it with the microfluidic chip base to form a cell culture chamber; (S3) Three-dimensional embedding: The cell spheres obtained in step (S1) are mixed with pre-cooled liquid basement membrane extract and solidified to form a 3D cell sphere construct; (S4) Dynamic culture: The 3D cell sphere construct obtained in step (S3) is transferred to the cell culture chamber of the chip obtained in step (S2), culture medium is added, and the dynamic perfusion system is started for continuous perfusion culture until the cell sphere forms polar structural features and MRP2 and BSEP are functionally expressed.

[0077] In a specific implementation, the following steps are included: (S1) Cells were cultured in a low-adsorption plate to form uniformly sized cell spheroids; (S2) A porous membrane is attached to the bottom of the cell culture chamber and assembled with the microfluidic chip base; (S3) After the cell spheres obtained in step (S1) are mixed and solidified with liquid extracellular matrix, they are injected into the culture chamber of step (S2) to form a 3D cell sphere construct; (S4) Start the dynamic perfusion system and perform continuous perfusion culture under physiological fluid shear force until the 3D cell spheroids form hepatocyte characteristic polarity and have transporter phenotype characteristics.

[0078] Applications of the liver chip platform of the present invention This invention provides the use of the 3D liver chip platform for evaluating the effects of drugs on transporter function.

[0079] Furthermore, the evaluation includes examining the effects of the drug on the expression, subcellular localization, and efflux activity of MRP2 and / or BSEP. Specific methods include, but are not limited to: The efflux function of MRP2 was assessed using the fluorescent substrate CDFDA, and the degree of inhibition of MRP2 function was reflected by detecting the intracellular CDF fluorescence intensity. The membrane localization integrity of MRP2 / BSEP proteins was assessed by immunofluorescence staining.

[0080] This invention provides the use of the aforementioned dynamic 3D liver chip platform for assessing the risk of drug-induced liver injury mediated by drug-induced transporters.

[0081] Furthermore, the assessment includes the simultaneous detection of multiple toxic phenotypes of the drug on hepatocytes, wherein the multiple toxic phenotypes are selected from one or more of the following to evaluate hepatocyte function and metabolism: transporter (MRP2 / BSEP) dysfunction, decreased mitochondrial membrane potential, changes in intracellular ATP content, and changes in CYP3A4 enzyme activity.

[0082] Furthermore, the assessment can distinguish between acute high-concentration effects and chronic low-concentration cumulative effects of drugs, and can be used to identify drugs with delayed-onset transporter inhibition toxicity risks.

[0083] This invention provides a method for screening or assessing the transporter-related drug delivery risk (DILI) of candidate drugs using the aforementioned dynamic 3D liver chip platform.

[0084] The main advantages of this invention include: (a) This invention utilizes widely available tumor cell lines (such as HepG2) for 3D cell spheroid culture, eliminating the need for expensive differentiation-inducing reagents, cytokines, or complex reprogramming procedures. This significantly reduces the cost per experiment, enabling highly reproducible, large-scale, high-throughput drug screening and DILI risk assessment, making it particularly suitable for the economic budgets of early-stage drug development.

[0085] (b) Commercial tumor cell lines are immortalized cells with stable genetic backgrounds and consistent proliferation characteristics, exhibiting minimal performance differences between different batches. Therefore, this platform can provide highly reproducible experimental data, facilitating cross-laboratory and cross-time comparisons and validations, and meeting regulatory requirements for model consistency.

[0086] (c) Dynamic 3D microfluidic chips obtained through specific preparation methods can induce tumor cell lines to upregulate the expression and function of key drug transporters (such as MRP2), forming a stable "transporter phenotype". This phenotype is easier to maintain during passage and long-term culture, thereby enabling accurate and predictable assessment of drug uptake, efflux and liver injury risks.

[0087] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0088] Example 1: Construction and functional verification of dynamic 3D liver chip platform.

[0089] Chip fabrication: Using CNC machining technology, an integrated perfusion channel is fabricated from optically transparent polycarbonate (PC) material. Figure 1 B) and removable culture chambers ( Figure 1 A) Figure 1 The microfluidic chip body (A shows the front view and top view on the left and right, respectively). A 20 µm PETE porous membrane is attached to the bottom of the culture chamber. Figure 1 A).

[0090] Cell spheroid preparation: Cells (such as HepaG2, HepaRG, Huh-7 cells, and organoids, etc.) (specifically, Huh-7 cells were used) were prepared at a concentration of 1.0 × 10⁻⁶. 4 Up to 5.0×10 6 Cells were seeded at a low-adsorption AggreWell™ 400 microplate at a density suitable for their microenvironmental properties in DMEM medium containing 10% FBS, 1% penicillin and streptomycin, and 1% Matrigel. After overnight culture, uniform 3D cell spheroids with a diameter of approximately 10-200 µm were formed.

[0091] 3D spheroid embedding and chip assembly: Cell spheroids were gently collected and resuspended in BME (BME and pre-cooled culture medium (components as described above) were mixed at a 2:1 ratio). 15 µL and 10 µL of the spheroid-BME suspension were added sequentially, layered, and allowed to solidify to form a stable 3D spheroid culture embedded in BME. This was then transferred to a cell culture chamber, and a microfluidic chip base was inserted to assemble the complete chip. Figure 1 C and Figure 1 D).

[0092] Dynamic perfusion culture: The perfusion channels of the chip are connected to a peristaltic pump via silicone tubing, with a flow rate of 0.5-1.0 dyn / cm. 2 The shear force was used for continuous perfusion culture. The culture medium was replaced with fresh medium every 24 hours.

[0093] With the same addition, a traditional 2D single-layer chip was constructed as a control.

[0094] Functional validation: On day 7 of culture, immunofluorescence assay, transporter function assay, cell viability assay, and CYP3A4 activity assay were performed.

[0095] Immunofluorescence results as follows Figure 2 A, Figure 2 B and Figure 2 As shown in C, after staining the 3D spheres and 2D cells on the chip with MRP2, BSEP and tight junction protein (ZO-1), MRP2 and BSEP in the 3D spheres were clearly located on the outer membrane of the cells and co-located with ZO-1, which is consistent with the polar localization characteristics of liver transporters in vivo; while in the 2D cells, the two transporters were diffusely distributed in the cytoplasm. The results of transporter function are as follows Figure 2 D and Figure 2 As shown in Figure E, the intracellular CDF fluorescence intensity was detected after incubation with the MRP2-specific fluorescent substrate CDFDA. The results showed that 3D spheroids could rapidly efflux CDF after 7 days of culture, with extremely low intracellular fluorescence; while 2D cells accumulated a large amount of CDF, indicating that they lacked functional MRP2.

[0096] The results of cell viability are as follows Figure 2 F and Figure 2 As shown in G, the changes in mitochondrial membrane potential and intracellular ATP content levels of 3D spheroids and 2D cells during continuous perfusion culture were measured. The results showed that 3D spheroids maintained cell viability for a longer period of time during culture, supporting long-term culture.

[0097] The results of CYP3A4 activity detection are as follows: Figure 2 As shown in H, the CYP3A4 enzyme activity was detected using a kit. The results showed that the CYP3A4 activity of 3D spheres was significantly increased by about 2.3 times on day 5 compared with day 2, while the activity of 2D cells was not increased and even decreased to below the detection limit.

[0098] The above results demonstrate that the dynamic 3D liver chip platform of the present invention has successfully constructed and realized the high expression and polarity distribution characteristics of functional transporters.

[0099] Example 2: Application of the platform to evaluate troglitazone, a known hepatotoxic drug.

[0100] Troglitazone, the first approved thiazolidinedione insulin sensitizer, exhibited significant hepatotoxicity, including jaundice, fatigue, and weakness. It was withdrawn from the market after only three years due to severe liver failure and even death. While the active ingredient of troglitazone has weak inhibitory efficacy against BSEP, its sulfate metabolites inhibit bile salt excretion. To verify the detection performance of the dynamic 3D liver chip of this invention, the 3D liver chip constructed in Example 1 and the 2D control chip were treated with 0, 20, 50, and 100 µM troglitazone, respectively.

[0101] The results showed that in the 3D chip, troglitazone reduced the membrane localization integrity of MRP2 in a concentration-dependent manner and led to a significant increase in intracellular CDF fluorescence intensity. Figure 3 A and Figure 3 C), indicating that MRP2 efflux function is inhibited. Simultaneously, TMRM staining showed a concentration-dependent decrease in mitochondrial membrane potential (C). Figure 3 E). However, in 2D chips, different concentrations of troglitazone all resulted in high CDF accumulation, with no significant difference (E). Figure 3 B and Figure 3 D), the mitochondrial membrane potential decreases significantly even at low concentrations ( Figure 3 E).

[0102] The results of this embodiment demonstrate that the platform of the present invention can accurately assess drug-induced multi-level transporter-mediated DILI with high sensitivity and a wide detection concentration window.

[0103] Example 3: Prospective assessment of transporter-related DILI risk of the novel drug omaglione using an application platform.

[0104] Oglitazone (OFG) is also an insulin sensitizer, and cases of elevated bilirubin levels in subjects have been reported during Phase II clinical trials. 3D liver microarrays that had been cultured for 7 days and possessed mature transport characteristics were treated with 0, 1, 10, 50, 100, and 200 µM oxalizone, i.e., acute exposure for 24 hours by applying 0, 1, 10, 50, 100, and 200 µM oxalizone to the flowing culture medium of a dynamic 3D liver microarray.

[0105] The results are as follows Figure 4 A and Figure 4 As shown in Figure B, intracellular CDF accumulation was observed only at concentrations above 50 µM, while significant CDF accumulation was observed at high concentrations of 100 µM and above.

[0106] Furthermore, 3D liver chips were treated with 0, 10, and 100 µM oxaliplatin and chronically exposed for 7 days to test their ability to detect the chronic low-concentration accumulation of the drug.

[0107] The results are as follows Figure 4 C Figure 4 D and Figure 4 As shown in E, significant and dose-dependent CDF accumulation was observed even at low concentrations, without a significant decrease in ATP content. Simultaneously, a significant decrease in mitochondrial membrane potential was also observed. Figure 4 (F), but the decrease was disproportionate to the amount of CDF accumulation, suggesting that there may be other cell damage mechanisms.

[0108] The results show that the dynamic 3D liver chip of the present invention successfully predicted the potential, time-dependent transporter-mediated risk of omaglione in DILI, which provides a reasonable mechanistic explanation for the sporadic hyperbilirubinemia observed in clinical trials.

[0109] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for safety screening of candidate drugs to obtain potential drugs with hepatoprotective properties, characterized in that, The method includes the following steps: (S1) A drug-induced liver injury risk assessment device is provided, the device comprising: a microfluidic chip and a power unit; the microfluidic chip comprises: a cell culture chamber and a flow path plate containing perfusion channels; in, When the cell culture chamber is embedded in the flow path plate, the cell culture chamber is located above the perfusion channel, and the cell culture chamber and the perfusion channel are separated by a porous membrane structure; the porous membrane allows liquid exchange between the cell culture chamber and the perfusion channel; The inlet and outlet of the perfusion channel in the flow path plate are connected by an external pipeline connected to a power device. The external pipeline and the perfusion channel inside the microfluidic chip together form a circulation path for flow perfusion culture, thereby realizing flow perfusion culture of cells in the cell culture chamber. in, The cell culture chamber contains a 3D cell structure formed from hepatocytes; The hepatocytes include: liver cancer cells; The levels of liver transport proteins in the hepatocytes were significantly increased; wherein the liver transport proteins include: MRP2, BSEP, or a combination thereof; (S2) In the experimental group, cells were cultured in a cell culture chamber in the presence of the candidate drug; in the control group, cells were cultured in a cell culture chamber under the same conditions but in the absence of the candidate drug. (S3) The concentrations (C1) of hepatic transporter proteins in the cells of the experimental group and (C2) of hepatic transporter proteins in the cells of the control group were detected and compared. If C1 was significantly less than C2, it indicated that the candidate drug had a risk of liver injury; otherwise, it indicated that the candidate drug was a potential drug with hepatoprotective properties; and / or The concentrations of specific substrates of liver transport proteins in the cells of the experimental group (D1) and the concentrations of specific substrates of liver transport proteins in the cells of the control group (D2) were measured and compared. If D1 was significantly greater than D2, it was suggested that the candidate drug had a risk of liver damage; otherwise, it was suggested that the candidate drug was a potential drug with liver safety. The liver injury risk mentioned above refers to the risk of drug-induced liver injury mediated by transporters.

2. The method as described in claim 1, characterized in that, The candidate drugs include: candidate insulin sensitizers.

3. The method as described in claim 1, characterized in that, The risks of liver damage include: cholestasis, mitochondrial damage, oxidative stress, endoplasmic reticulum stress, or a combination thereof.

4. The method as described in claim 1, characterized in that, Step (S3) further includes: The mitochondrial membrane potential A1 of the cells in the experimental group and the mitochondrial membrane potential A2 of the cells in the control group were detected and compared. If there was no significant difference between A1 and A2, it would suggest that the candidate drug is a potential drug with liver safety.

5. A microfluidic chip for drug-induced liver injury risk assessment, characterized in that, The microfluidic chip includes: a cell culture chamber and a flow path plate containing perfusion channels; in, When the cell culture chamber is embedded in the flow path plate, the cell culture chamber is located above the perfusion channel, and the cell culture chamber and the perfusion channel are separated by a porous membrane structure; the porous membrane allows liquid exchange between the cell culture chamber and the perfusion channel; The irrigation channel includes an inlet and an outlet; in, The cell culture chamber contains a 3D cell structure formed from hepatocytes; The liver cells include liver cancer cells.

6. A drug-induced liver injury risk assessment device, characterized in that, The device comprises: the microfluidic chip and power unit as described in claim 5; The inlet and outlet of the irrigation channel in the flow path plate are connected by an external pipeline connected to a power device. The external tubing and the internal perfusion channels of the microfluidic chip together form a circulation path for flow perfusion culture.

7. The use of the microfluidic chip of claim 5 or the device of claim 6, characterized in that, Used for: (a) Screening for potential drugs with hepatoprotective properties; and / or (b) Assess the risk of liver injury from candidate drugs in vitro; (c) Differentiate the acute hepatotoxic effects of drugs from the cumulative effects of chronic hepatotoxicity in vitro; (d) Evaluate in vitro the effects of the drug on the expression levels, subcellular localization, and efflux activity of MRP2 and / or BSEP.

8. The use as described in claim 7, characterized in that, The liver injury mentioned includes: acute liver injury and chronic liver injury.

9. A method for preparing the microfluidic chip according to claim 5, characterized in that, The method includes the following steps: (I) Provide a cell culture chamber and a flow path plate containing perfusion channels; The irrigation channel includes an inlet and an outlet; When the cell culture chamber is embedded in the flow path plate, the cell culture chamber is located above the perfusion channel, and the cell culture chamber and the perfusion channel are separated by a porous membrane structure; the porous membrane allows liquid exchange between the cell culture chamber and the perfusion channel; (II) Provides a 3D cellular structure formed from hepatocytes, The hepatocytes include: liver cancer cell lines; (III) The 3D cell structure provided in step (II) is transferred to the cell culture chamber provided in step (I) to obtain the microfluidic chip of claim 5.

10. The method as described in claim 9, characterized in that, Step (II) includes the following sub-steps: (II-1) The hepatocytes were seeded into microplates with low adsorption and cultured to form 3D cell structures; (II-2) Collect the 3D cell structure and disperse it in a premix of extracellular matrix and pre-cooled culture medium to obtain an embedded 3D cell structure, thereby providing a 3D cell structure formed by hepatocytes.

Images